Studies of the photosensitized oxidation have demonstrated that photodynamic oxidation of methionine is mediated by singlet oxygen ((1)O(2)). In this study, we demonstrated that phagocytosing human polymorphonuclear leukocytes (PMN), but not resting PMN, oxidized both intracellular and extracellular methionine to methionine sulfoxide. N-ethylmaleimide, which inhibits phagocytosis and cellular metabolism, inhibited the oxidation of methionine. Neutrophils from patients with chronic granulomatous disease did not oxidize methionine even in the presence of phagocytosis. The oxidation of methionine by phagocytosing normal PMN was inhibited by (1)O(2) quenchers, (1.4-diazabicyclo-[2,2,2]-octane, tryptophan, NaN(3)), myeloperoxidase (MPO) inhibitors (NaN(3), KCN) and catalase. In contrast, superoxide dismutase, ethanol, and mannitol had no effect. Furthermore, (1)O(2) quenchers did not interfere with the production of superoxide (O(2) (-)) by phagocytosing PMN. The combination of catalase and SOD did not enhance the inhibition of methionine by phagocytosing PMN. On the other hand, deuterium oxide stimulated the oxidation of methionine by PMN almost 200%.H(2)O(2) at high concentrations oxidized methionine to methionine sulfoxide. However, when similar amounts of H(2)O(2) were added to human PMN, they did not oxidize methionine. In contrast, when H(2)O(2), at concentrations too low to oxidize methionine by itself, was added to the granular fraction, but not the soluble fraction, they oxidized methionine to methionine sulfoxide. The oxidation of methionine by the combination of H(2)O(2) and granular fractions was inhibited by (1)O(2) quenchers and MPO inhibitors, but it was stimulated by deuterium oxide. Removal of chloride anion also prevented the oxidation of methionine by the granular fractions. Our results suggest that the oxidation of methionine by phagocytosing PMN is dependent on the MPO-mediated antimicrobial system (MPO-H(2)O(2)-Cl(-)). They also suggest, but do not prove that the oxidation of methionine is mediated by (1)O(2). Oxidation of methionine may be one of the mechanisms that human PMN damage microorganisms.